A scoping review: What are the cellular mechanisms that drive the allergic inflammatory response to fungal allergens in the lung epithelium?

Abstract Allergic airway disease (AAD) is a collective term for respiratory disorders that can be exacerbated upon exposure to airborne allergens. The contribution of fungal allergens to AAD has become well established over recent years. We conducted a comprehensive review of the literature using Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines to better understand the mechanisms involved in the allergic response to fungi in airway epithelia, identify knowledge gaps and make recommendations for future research. The search resulted in 61 studies for final analysis. Despite heterogeneity in the models and methods used, we identified major pathways involved in fungal allergy. These included the activation of protease‐activated receptor 2, the EGFR pathway, adenosine triphosphate and purinergic receptor‐dependent release of IL33, and oxidative stress, which drove mucin expression and goblet cell metaplasia, Th2 cytokine production, reduced barrier integrity, eosinophil recruitment, and airway hyperresponsiveness. However, there were several knowledge gaps and therefore we recommend future research should focus on the use of more physiologically relevant methods to directly compare key allergenic fungal species, clarify specific mechanisms of fungal allergy, and assess the fungal allergy in disease models. This will inform disease management and future interventions, ultimately reducing the burden of disease.

Thermotolerant genera such as Aspergillus and Penicillium may exert their allergenic effects through colonisation of the airway, whilst non-thermotolerant fungi such as Alternaria may still cause an allergic response through inhalational exposure and sensitisation. 11,12 Fungi have specific features that are recognisable to the human immune system, termed pathogen-associated molecular patterns (PAMPs) and include β-glucan, chitin and mannans. In addition, fungi produce proteases and glycosidases to degrade material for nutrient release, which can also degrade epithelial tight junctions in the lung and induce allergic responses. 1,4,11,13

| Allergic airway disease
Allergic airway disease (AAD) is a collective term for respiratory disorders that can be exacerbated upon exposure to airborne allergens, including fungal agents, pollen and house dust mite (HDM). 11 These disorders include asthma, chronic rhinosinusitis (CRS) and allergic bronchial pulmonary aspergillosis (ABPA), which affect millions of people globally. 1,[14][15][16][17] Typically, AAD is defined as an allergic response in individuals characterised by increased serum IgE, airway inflammation, airway remodelling (including peribronchial fibrosis and increased collagen deposition), increased type 2 cytokine profile (e.g. Interleukin (IL) 4, IL5, IL13, IL25, IL33, thymic stromal lymphopoietin (TSLP)), airway eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (AHR). 1,13,18 Upon inhalation, the airway epithelium is the first cellular barrier that fungal allergens encounter and thus has important protective functions, including a physical barrier to inhaled particles and microbes, immunological functions and mucociliary clearance. [18][19][20][21] Allergens can become trapped in the nasal cavity, trachea, bronchi, small airways, and alveoli 5,22,23 depending on their size, with the majority of particles being deposited within the nasal cavity and upper airways (due to size and turbination of the air), and some smaller particles (<5 μm) penetrating into the smaller airways. 4 The upper airway has a columnar pseudostratified epithelium made up of cilia, goblet cells and/or club cells and basal cells, 20,21 whilst the smaller airways and terminal bronchioles are more cuboidal. 24 Various structural and functional differences exist between the large and small airways. 24 Goblet cells produce mucus, which in combination with the cilia, trap particles that impact the epithelium and move them up the airway to the mouth via the mucociliary escalator. 20 Pattern recognition receptors (PRRs) on the epithelium can detect fungal allergens/PAMPs or danger associated molecular patterns (DAMPs), triggering signalling cascades that induce the release of several cytokines and chemokines 14,19,21,25 that drive allergic responses.
T A B L E 1 Known thermotolerant a and non-thermotolerant fungal allergens. [7][8][9][10]  Where there is evidence that spores are present in both environments, the predominant environment is listed first.
The prevalence of allergic respiratory conditions has increased over the last few decades 4,26 and fungal allergens can play a significant role in the development of AAD 4 with several species of fungi also recognised as 'sensitisers'. 1,3,27 Sensitisation refers to the process by which the body becomes abnormally sensitive to an allergen through repeated exposures and is the first stage in the development of allergy. 28 Repeated inhalation of fungal allergens can contribute to the development and exacerbations of asthma and allergic rhinitis. 29 In particular, exacerbations of allergic asthma have been linked with fungal spore exposure 4,29 and there is a correlation between severe asthma and fungal sensitisation 17 with 35%-75% of severe asthmatics having some form of fungal sensitisation (compared to the general population with a rate of 3%-10%). 7 million people globally suffer from severe asthma with fungal sensitisation. 29 Understanding the biological processes driving the contribution of fungal allergens to AAD is essential for improving disease management, treatment strategies, interventions, and ultimately reducing the burden of disease.

| Aim
This review aimed to collate the current understanding of mechanisms involved in the allergic response to fungi in the airway epithelium, identify knowledge gaps and make recommendations for future research.

| Search strategy
A comprehensive Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)-based literature search was conducted using the PubMed database in January 2022. The search string comprised fungal, allergy, respiratory and epithelial/macrophage search terms (Supporting Information S1).

| Study selection
The selection of studies occurred in three stages: first by titles, then by abstracts and then by full text. All titles and abstracts were reviewed independently by two reviewers. Table 2 details the inclusion and exclusion criteria. Only data from relevant studies within the last 15 years were extracted at the full text stage to focus on the most current literature and avoid repetition of information. However, these papers were checked for any relevant data and referenced in the discussion where appropriate.

| Data extraction
Using a pre-designed template, the following study data were extracted: author(s) and year, fungal allergens, potential target mechanisms, experimental models, and experimental outcomes.
Only data relating to fungal allergens and epithelial cells were extracted. Other exposure compounds or cell types were noted when relevant to fungal allergen activity (either for confirmation of allergic models or when related to events surrounding the epithelium). Potential target mechanisms were defined as any molecule or pathway that was either (a) the specific focus of the study, (b) a molecule or pathway that was further validated using knockouts (KOs), inhibitors or gene silencing/overexpressing techniques, or (c) an endpoint molecule where pathways or genes were manipulated to observe the effects on said molecule. Molecules that were investigated using appropriate validation techniques but found not to be involved in specific pathways were still included as potential target mechanisms. Molecules that were used as endpoints to confirm allergic responses in models were not included as potential target mechanisms unless they underwent further investigation.

| Quality scoring
A quality-scoring tool was adapted from a previous review. 27

| Overview of included studies
The search string provided 440 results from the PubMed database.
Study selection according to PRISMA guidelines is outlined in Figure 1, which resulted in 61 studies for data extraction (Table 3).
See Supporting Information S3 for further details of each study.

| Models
Of the 61 studies, 24 (38%) were performed in vitro, 16 (27%) in vivo and 21 (35%) used both in vivo and in vitro models. An overview of in vitro models (n = 45) is described in Figure 2A. In vivo studies (n = 37) predominantly used mouse models, with just a single study 49 using a rat model. The induction of allergy methods in in vivo models are described in Figure 2B,C. Whilst all in vivo models used allergen exposure compared to a control to model AAD in some way, there was large heterogeneity in both the methods of sensitisation and challenge applied and the disease-related outcomes investigated.
A range of different fungal species were used in the selected studies with Aspergillus (n = 41) and Alternaria (n = 31) being the most studied fungal genera. Fungal allergens were applied in various forms, including culture filtrate or extract (CFE), conidia and purified proteases. The breakdown of fungal allergens can be found in Figure 2D. Only one study compared conidia and CFE

| Potential mechanisms of fungal-induced allergy
In total, 120 potential target mechanisms (individual molecules or pathways) were identified, 65 (54%) were identified in only one study (Supporting Information S4). Of the 54 (44%) targets identified in more than one study, 17 (28%) of these were identified in five or more studies (Figure 3), giving a greater confidence that these potential target mechanisms are involved in the allergic response to fungi. Proteases were the most investigated target (n = 19, 31%), followed by IL33 (n = 18, 30%), calcium signalling, and protease-activated receptor 2 (PAR2) (n = 13, 21% each).

| Quality assessment
An overview of how studies scored in each domain is shown in  Penicillium citrinum protease Pen c 13, normal and denatured � Pen c 13 exposed mice showed greater AHR, marked GCM, and increased BALF leukocytes and total IgE versus controls � Pen c 13 exposed mice showed increased collagen deposition and elevated lung hydroxyproline versus controls � Pen c c13 treatment resulted in time-dependent cleavage of occludin and E-cadherin. ZO-1 was markedly reduced (Continues) Penicillium chrysogenum, crude antigen preparation (PCE) � NGF levels were increased in BALF and serum of mice exposed to 50 and 70 μg PCE at D0 and D1 versus controls. Single exposure did not increase NGF � NT4 levels increased in BALF and serum of mice exposed to 50 and 70 μg PCE versus controls. Single exposure did not increase NT4 � NT3 levels increased in BALF of mice exposed to 50 and 70 μg PCE versus controls. Single exposure did not increase NT3 � There was no change in BDNF levels between PCEtreated mice versus control A. alternata extract � AA exposure caused prominent airway inflammation, increased eosinophils, membrane thickening, increased mucus production and increased airway remodelling � RNA-seq: 403 upregulated genes and 108 downregulated genes after 6 weeks exposure � ST2 expression was increased in AA exposure, IL33 was marginally decreased versus controls � AA downregulated keratinisation in epithelial cells and upregulated genes relating to Ig expression and receptors as well as serine proteases such as Tpsb2 and Tmprss2 and eosinophil migration � NLRP3 was significantly increased in patients with ABPA and to a lesser extent, IPF. NLRP3, caspase 1 and ASC increased and co-localised in AF-exposed mice and in AFexposed epithelial cells, which was reduced by PI3K-δ inhibitors � IL1β inhibition improved AF induced allergic lung inflammation, including reductions in infiltrating cells � AA-induced allergic inflammation: NLRP3 and PI3K-δ inhibitors reduced increases in eosinophils in BALF, Th2 cytokines (IL4, IL5, IL13) and IL1β in lungs of mice � NLRP3 inflammasome assembly and activation was increased in the lungs of AF-and AA-exposed mice � PI3K-δ played a role in NLRP3 inflammasome assembly and activation in bronchial epithelial cells A. fumigatus crude antigen extract � AF exposure: Increased BALF cell numbers (especially eosinophils), IgE, Th2 cytokines and AHR. Th2 cytokines were reduced by NF-κB inhibitors � ER stress was increased in lungs of AF-exposed mice � GRP78 was also upregulated in lungs of ABPA patients, and GRP78 and CHOP were upregulated in AF-exposed mice � ER stress may be involved in pathogenesis of AF-related allergic lung disorders, ER stress involved PI3K-δ and mtROS generation Leino et al., 2013 67 A. alternata, Cladosporium herbarum, culture extracts � Significant dose-dependent decrease in TEER in polarised cultures observed 1 h post challenge with AA but recovered quickly at lower doses. Higher dose of AA, TEER remained lower at 24 h. CH did not affect TEER. In ALI cultures, AA had no effect on barrier function in healthy donors but severe asthmatics saw a dosedependent decrease within 3 h but recovered by 24 h � AA significantly induced release of inflammatory cytokines and increased permeability of polarised epithelial cells potentially due to serine and aspartate protease activity � Differentiated primary cells had a blunted IL8 response but those from asthmatics were more susceptible to barrier weakening � Cells stimulated with AA showed rapid increase in cytosolic free Ca 2+ , observed after 200 s and peaked between 400 and 600 s � Only AA induced IL6, IL8 and GM-CSF production versus other fungi. AA did not induce eotaxin, eotaxin-2 or RANTES. HI-AA did not induce cytokine production � Aspartate protease inhibitors inhibited cytokine production and Ca 2+ response in AA exposed epithelial cells � Aspartate proteases but not serine proteases in AA activated PAR2 in epithelial cells � AA but not other fungal extracts activated IL6 production/ release A. alternata extract � AA-induced IL33 release decreased by inhibition of VDAC1 in a concentration-dependent manner � Inhibitors and silencing of VDAC1 reduced AA-induced release of ATP and Ca 2+ . VDAC1 was involved in ATP release in epithelial cells exposed to AA � Reduced cholesterol inhibited initial ATP release.
Cholesterol played a role in ATP release and Ca 2+ uptake in AECs exposed to AA � Plasma membrane localisation of VDAC1 was dependent on cholesterol

F I G U R E 3
Potential target mechanisms identified in five or more studies. Potential target mechanisms were defined as any molecule or pathway that was either (a) the specific focus of the study, (b) a molecule or pathway that was further validated using knockouts, inhibitors or gene silencing/overexpressing techniques, or (c) an endpoint molecule where pathways or genes were manipulated to observe the effects on said molecule. Molecules that were investigated using appropriate validation techniques but found not to be involved in specific pathways were still included as potential target mechanisms. Molecules that were used as endpoints to confirm allergic responses in models were not included as potential target mechanisms unless they underwent further investigation. ATP, adenosine triphosphate; Ca 2+ , calcium; EGFR, epidermal growth factor receptor; IL, interleukin; PAR, protease-activated receptor; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; TSLP, thymic stromal lymphopoietin.
Mice do not develop allergic airway disease spontaneously, so they must be artificially induced and are often done so in two phases (sensitisation then challenge), representative of the allergic immune response seen in humans. 94 Therefore, sensitisation to a fungal allergen followed by subsequent challenge(s) with the same fungal allergen may be regarded as the most representative model of fungal allergy development. In addition, inhalation or intranasal instillation of allergens would also best mimic human exposure to airborne fungal allergens 94,95 and should be taken into account when developing in vivo models for AAD.

| Different fungi/fungal components
The few studies (n = 9, 15%) that compared different fungal species suggest that they can induce different responses in airway epithelia.  Different components of the same fungi also induced diverse responses. Immortalised mouse lung epithelial cells exposed to both heat-inactivated and live Aspergillus conidia for 6, 24 and 48 h showed increased IL25, IL33 and TSLP compared to controls. 60 This is in direct contrast to the results described above where Aspergillus extract did not induce IL25, IL33 or TSLP in either primary HNE or mouse lung epithelial cells. 73,85,88 This suggests that epithelial cells have diverse responses to different fungi and fungal components. Unfortunately, the heterogeneity between studies and lack of studies directly comparing these

| Potential mechanisms of fungal-induced allergy
By collating and comparing the data extracted from the 61 studies, potential mechanisms within airway epithelial cells that drive fungalinduced allergic responses (Figure 3) have been identified. However, it should be noted that many of the studies within this review used fungal CFE as an exposure compound in contrast to spores, which may not be a physiologically relevant method of exposure for humans on a day-to-day basis. 16 Epithelial barrier disruption is a key mechanism in AAD and has been identified as a target mechanism. As many reviews have been It should be noted, however, that inhibiting PAR2 in HNE and HBE cells did not completely reduce intracellular calcium or ATP release, cytokine or mucin secretion, or inflammation, suggesting additional PAR2-independent mechanism(s) for allergic responses. 45,74,81,91 This is supported by recent studies showing non-protease activation of the MAPK pathway, 98 and attenuated not abrogated responses in PARdeficient mice. 13

| EGFR pathway
A role for the epidermal growth factor receptor (EGFR) pathway in fungal allergy was supported in 6 (10%) of the studies. 45,49,55,75,81,91 The EGFR pathway plays a role in mucin production, airway epithelial repair and airway remodelling in response to disruption or injury to the epithelial layer. [102][103][104][105] Activation of EGFR through fungal allergens increased expression of MUC5AC and mucus production, 75 EGFR is a transmembrane tyrosine kinase that is activated by several ligands, including epidermal growth factor (EGF), heparinbinding EGF (hb-EGF), transforming growth factor α (TGF-α) and amphiregulin. 102,103,111 An increased level of both EGF and TGF-α was observed in the bronchoalveolar lavage fluid (BALF) of rats exposed to  105,111 While activation of the MAPK pathway through EGFR phosphorylation has been implicated in mucin production in asthma 111 and in response to cigarette smoke exposure, 108 the mechanism appears more complex in fungal-induced allergy. EGFR activity was required for mucin expression in lung carcinoma cells treated with A. fumigatus extract, but no increase in the level of phosphorylated EGFR was observed. 91 Instead, Aspergillus proteases induced increased mucin production by activating Ras and phosphorylating ERK, with ERK1/2 activation likely achieved through intracellular calcium flux. 91 The downstream effects of activation of signalling pathways PI3K and PLC are discussed in other sections. In addition, EGFR activation was associated with fungal-induced secretion of IL33 55,81 (see section below), and ATP has also been associated with activating EGFR in the wider literature. 109

| ATP and purinergic receptor-dependent release of IL33
IL33 was identified as the second most studied molecule in this review (n = 18, 30%). It is an epithelial-derived cytokine that plays an integral role in activating type 2 immunity in response to allergens. 112 IL33 was rapidly released upon exposure to Alternaria, reaching maximal levels within 2 h in HBE cells 55 and within 1 h in BALF in an Alternaria-exposed mouse model. 63 In addition, IL33 was released in response to PAR2 agonists, and both protease and PAR2 inhibitors reduced IL33 secretion, 81 suggesting that fungal protease-induced IL33 secretion may (at least in part) involve activation of PAR2 receptors. 11,113 IL33 was linked with ATP, 63 63,74 There is a strong evolutionary link between calcium and ATP signalling, 114 and these signalling molecules were identified in 13 (21%) and 9 (15%) studies, respectively.
ATP is known as a cellular energy store. However, it has also signalling functions within the inflammatory response as a DAMP and is released from the cell in response to cellular injury. 18,115,116 ATP production and release is induced by fungal proteases 11 and the use of serine and cysteine protease inhibitors reduced Alternaria-associated increases in ATP release from HBE cells. 74,77 ATP release across the cell membrane can occur through exocytosis and transmembrane channels. 116 In response to fungal allergens, ATP was released via both exocytosis 74 or VDAC1 in the apical membrane of HBE cells 86 ( Figure 5B).
ATP is part of purinergic signalling and upon release activates purinergic receptors on the cell surface. 116 driving specific characteristics of allergic inflammation (as described in Figure S1), 120,121 with some evidence that these cytokines were dependent on signal transducer and activator of transcription 6 (STAT6) signalling 47,48 and NF-κB. 66 124,126,129,130 Two studies looked at the role of fungal-induced oxidative stress in barrier integrity. 84 ( Figure 6B).
The ER stress markers GRP78 and CHOP were increased in mice 65,66 and murine tracheal cells exposed to Aspergillus extract. 66 These increases were reduced in the presence of a ROS scavenger, 66 suggesting that ROS may also play a role in inducing ER stress. Wider evidence suggests that PI3K signalling is involved in Type 2 immune responses, with the PI3K-δ subunit in particular mediating ROSdriven allergic inflammation and ER stress. 128,134 Evidence for a similar mechanism in fungal allergy was provided by two studies demonstrating increased PI3K-δ mRNA, ER stress markers and 24 of 32 impaired protein folding in murine tracheal cells following Aspergillus exposure that were reduced upon inhibition of PI3K-δ. 65,66 As well as reducing ER stress, inhibition of PI3K-δ also decreased Aspergillusinduced mtROS, protein oxidation, and membrane lipid peroxidation, which restored redox balance, and, in turn, reduced AHR, infiltration of eosinophils and mucin expression. 58,65,66 The same was seen when a ROS scavenger was used, 66 further confirming the role of PI3K-δ and mtROS in oxidative stress and the allergic response to fungi.
Allergen exposure can also affect ER and mitochondrial morphology, resulting in potential crosstalk between the ER and mitochondria. 124 In Aspergillus exposed mice, there was a decrease in membrane fluidity, and an aberrant morphology in the ER membrane as well as swollen mitochondria with a decreased distance between the two organelles. 65 Further investigation in mouse lung tissue confirmed that ER-associated ITPR transmembrane channels and the mitochondrial outer membrane channel VDAC1 became more closely associated in response to Aspergillus exposure. 65 Such sites of physical contact are known as mitochondria-associated ER membranes (MAMs) 127 ( Figure 6C) and ER-mitochondrial crosstalk may play a role in the allergic response to fungi. One hypothesis is that the movement of calcium between the ER and the mitochondria causes the release of ATP from the mitochondria into the cytosol. In support of this, calcium was released from the ER, uptake of calcium into the mitochondria increased and mitochondrial ATP levels were reduced upon exposure of HBE cells (BEAS-2B) to Aspergillus. 65 All of these Aspergillus-induced changes were reversed or reduced in the presence of PI3K-δ or ER stress inhibitors. 65 An overview of all the potential mechanisms driving fungalinduced allergy in the lung epithelium and how they interact can be found in Figure 7.

| Recommendations for future work
This review of the current literature, combined with the quality assessment designed to highlight the strengths and weaknesses of the included studies in understanding the cellular mechanisms that drive the allergic inflammatory response to fungal allergens in the lung epithelium, has identified several knowledge gaps, which the following recommendations aim to address:

| CONCLUSIONS
In summary, this review has brought together the current literature on fungal allergy in airway epithelia, identifying mechanisms including PAR2, EGFR, ATP and IL33 signalling, and oxidative stress.
These pathways can induce the classic hallmarks of allergy including increased mucus production, eosinophilia and AHR. Ambiguity remains, however, due to the heterogeneity of current models and